Inorganic polyphosphate (polyphosphate or PolyP) is a linear polymer of orthophosphate, linked by phosphoanhydride bonds1-3. It has been found in mammalian cells and lower organisms and localized to lysosomes, dense granules, mitochondria and nuclei. The polyphosphate polymer varies in length from cell to cell and in different organisms, ranging from 60-100 in human platelets, to thousands of phosphate units in some bacteria4-6. In platelets, abundant polyphosphate has been localized in dense granules7 and released upon activation, whereupon it has been found in platelet-rich thrombi at concentrations of 1-3 uM8. At physiologic pH, each internal unit has a monovalent negative charge, and thus the polymers are highly anionic. This property led to the finding that polyphosphate can provide a physiologic anionic surface on which factor XII, prekallikrein and high molecular weight kininogen, assemble for contact activation of coagulation9. Subsequent studies found that polyphosphate is prothrombotic and pro-inflammatory in in vivo mouse models8,10, acting at multiple steps in the coagulation cascade8,11-17. The effects of polyphosphate on coagulation have been found to be concentration- and size-dependent14. Thus, platelet-sized polyphosphate (P60-100) has been found to primarily accelerate thrombin-mediated activation of factor XI and factor V, while larger size polyphosphate has been found to trigger coagulation via contact activation of factor XII and enhances fibrin polymerization.
The complement system comprises over 30 soluble and membrane-bound proteins, contributing to innate and adaptive immunity, aiding in the disposal of danger-associated molecular patterns. Complement activation, which often occurs in concert with coagulation, is achieved via three pathways—the lectin pathway (LP), the classical pathway (CP) and the alternative pathway (AP). These converge with C3 convertase-mediated transformation of C3 into C3a and C3b. The C3a anaphylatoxin recruits leukocytes and activates platelets20. C3b deposition on bacteria promotes opsonization by leukocytes and is required for formation of the C5 convertase that cleaves C5 into C5a and C5b. C5b rapidly binds to C6, forming a tight C5b,6 complex, which then binds to C7, yielding the C5b-7 complex. This attaches to the outer leaflet of a target membrane. The subsequent addition of the heterotrimeric C8αβγ further stabilizes and anchors the now C5b-8 complex to the cell by inducing a conformational change in C8 and burying a hydrophobic tail through the lipid bilayer. Multiple C9 subunits finally join for assembly of the C5b-9 pore-like, lytic membrane attack complex (MAC)21.
C1-inh is a serine protease inhibitor (serpin) that circulates in the blood and is a major negative regulator of complement activation, interfering with the activation of the lectin and classical pathways by neutralizing MBL-associated serine protease (MASP)1 and C1s, respectively70,71. The effect of C1-inh on MASP1 and C1s is augmented by the presence of heparin72. PolyP has been shown to bind to C1-inh69.
Inappropriate complement activation has been implicated in inflammation, immune disorders, and in the pathology of many diseases or disorders.